The transcription factor OsWRKY10 inhibits phosphate uptake via suppressing OsPHT1;2 expression under phosphate-replete conditions in rice

Abstract Plants have evolved delicate systems for stimulating or inhibiting inorganic phosphate (Pi) uptake in response to the fluctuating Pi availability in soil. However, the negative regulators inhibiting Pi uptake at the transcriptional level are largely unexplored. Here, we functionally characterized a transcription factor in rice (Oryza sativa), OsWRKY10. OsWRKY10 encodes a nucleus-localized protein and showed preferential tissue localization. Knockout of OsWRKY10 led to increased Pi uptake and accumulation under Pi-replete conditions. In accordance with this phenotype, OsWRKY10 was transcriptionally induced by Pi, and a subset of PHOSPHATE TRANSPORTER 1 (PHT1) genes were up-regulated upon its mutation, suggesting that OsWRKY10 is a transcriptional repressor of Pi uptake. Moreover, rice plants expressing the OsWRKY10–VP16 fusion protein (a dominant transcriptional activator) accumulated even more Pi than oswrky10. Several lines of biochemical evidence demonstrated that OsWRKY10 directly suppressed OsPHT1;2 expression. Genetic analysis showed that OsPHT1;2 was responsible for the increased Pi accumulation in oswrky10. Furthermore, during Pi starvation, OsWRKY10 protein was degraded through the 26S proteasome. Altogether, the OsWRKY10–OsPHT1;2 module represents a crucial loop in the Pi signaling network in rice, inhibiting Pi uptake when there is ample Pi in the environment.


Introduction
Phosphorus (P) is one of the three major macronutrients indispensable for plant growth and development (Raghothama, 1999;Hawkesford et al., 2012). Although this element was first discovered in urine, P fertilizers are mainly derived from a fi-nite resource on the earth, namely phosphate (Pi) rock, which is estimated to be depleted in the near future (Van Kauwenbergh, 2010;Lambers and Plaxton, 2015). In the soils of some natural or agricultural ecosystem, the availability of Pi for plants is low. This paper is available online free of all access charges (see https://academic.oup.com/jxb/pages/openaccess for further details) For example, in both acidic and calcareous soils with suboptimal fertilization, Pi is precipitated by cations (e.g. Al 3+ /Fe 3+ in acidic soils and Ca 2+ in calcareous soils) and becomes unavailable for plants (Lambers and Plaxton, 2015). To cope with the scarcity of Pi in soil, plants have evolved a suite of delicate systems for the sensing, uptake, distribution, and metabolism of Pi (Gu et al., 2016;Huang et al., 2020;Y. Wang et al., 2021). During the past two decades, dozens of regulators have been reported to be involved in Pi starvation signaling (Ueda et al., 2021;Z.R. Wang et al., 2021;Lambers, 2022;Paz-Ares et al., 2022), among which a subclade of Myeloblastosis (MYB) transcription factors (TFs) termed PHOSPHATE STARVATION RESPONSE (PHR) have been demonstrated to be the central regulators activating the transcription of ~50% of Pi starvationinduced (PSI) genes (Bustos et al., 2010;Guo et al., 2015).
In addition to the post-translational regulation, transcriptional regulation represents another important checkpoint monitoring the abundance of these PHT1 genes. An increasing number of TFs have been demonstrated to directly regulate the expression of PHT1 genes (Gu et al., 2016, and references therein). In rice, OsPHT1;1 is a unique PHT1 member not transcriptionally responsive to Pi (Sun et al., 2012). In our recent work, we showed that the basal expression of OsPHT1;1 is maintained by a pair of WRKY TFs, OsWRKY21 and OsWRKY108 , demonstrating that transcriptional activation of PHT1 gene(s) is an integral part of the Pi signaling network under Pi-replete conditions. Nevertheless, several lines of preliminary evidence indicate that transcriptional suppression of PHT1(s) occurs simultaneously (Mukatira et al., 2001;Gu et al., 2017). Unlike OsPHT1;1, another rice PHT1 gene highly expressed under Pi-replete conditions, namely OsPHT1;2, is a direct target of OsPHR2 and is transcriptionally induced by Pi starvation (Ai et al., 2009;Liu et al., 2010;Secco et al., 2013;Zhang et al., 2021). In contrast, under Pi-replete conditions, OsPHT1;2 transcription is subjected to negative regulation, as evidenced by the fact that, in Pi-replete osmyb1 mutants, OsPHT1;2 expression is elevated to a level comparable with that under Pi-deficient conditions (Gu et al., 2017). Further experimental evidence in support of this presumption is that the promoter of a PHT1 gene in Arabidopsis, AtPT2/AtPHT1;4, is bound by an unknown nuclear protein factor from Pi-sufficient plants (Mukatira et al., 2001). In spite of these findings, the TFs negatively regulating PHT1 genes by binding to their promoters are largely unexplored.
In the present study, a WRKY TF in rice (OsWRKY10; WRKY10 hereafter), was functionally characterized regarding its role in maintaining P homeostasis. WRKY10 was found to inhibit Pi uptake under Pi-replete condition by suppressing OsPHT1;2 expression via direct binding to its promoter. Thus, we established that the WRKY10-OsPHT1;2 module in rice is important for inhibiting Pi uptake from Pi-replete environments.

Plant materials, vector construction, rice transformation, and growth conditions
All rice (Oryza sativa) materials used in this study were Nipponbare cultivar. Mutants of pht1;2 and wrky10 were generated using the CRISPR/ Cas9 system (Miao et al., 2013). Spacers residing in exons of each gene were selected from the website (Xie et al., 2017). These spacers were sequentially ligated into the intermediate vector pOs-sgRNA and the expression vector pH-Ubi-cas9-7 as described in Zhang et al. (2021). The wrky10 mutant was crossed with the pht1;2 or myb1 mutant, and the F 2 generation of wrky10 pht1;2 or wrky10 myb1 double mutants were used for experiments. The primers used to identify WRKY10, PHT1;2, and MYB1 are listed in Supplementary Table S1. For overexpression of WRKY10, the double cauliflower mosaic virus 35S promoter and NOS terminator were subcloned into the vector pCAMBIA1305.1-GUSPlus, and the new expression vector was named pCAMBIA1305.1-2 × 35ST. The full-length ORF of WRKY10 was amplified from the Nipponbare cDNA and then ligated to pCAM-BIA1305.1-2 × 35ST.
For tissue localization, the GUSPlus gene and NOS terminator were subcloned into the vector pCAMBIA1300, and the new vector was named pCAMBIA1300-GN. The 2195 bp DNA fragment upstream of the start codon of WRKY10 was amplified from Nipponbare genomic DNA and cloned into pCAMBIA1300-GN.
For ChIP-qPCR, the promoter of the rice Actin1 gene, 3×FLAG, NOS terminator, and the promoter of maize ubiquitin were subcloned into the vector pCAMBIA1305.1-2 × 35ST, and the new expression vector was named pCAMBIA1305-AFU. Then the full-length ORF of WRKY10 without a stop codon was ligated to pCAMBIA1305-AFU.
For transcriptional activator vectors, the VP16 sequence was synthesized by Genscript according to the methods described by Kong et al. (2016), and was then cloned into a WRKY10 overexpression vector using the ClonExpress II One Step Cloning Kit (Vazyme).
The above constructs were transformed into callus dedifferentiated from the mature embryo of Nipponbare via Agrobacterium tumefaciensmediated japonica rice transformation as described in Jia et al. (2011). All the primers using for vector construction are listed in Supplementary  Table S1.
For hydroponic culture experiments, the rice seeds were disinfected with 30% NaClO and placed in an incubator at 30 °C for 2 d, then transferred to a plastic net floating on 0.5 mM CaCl 2 solution. After 3 d, the seedlings were transferred to Yoshida solution (Yoshida, 1976) or 1/2 strength Kimura B solution (Yamaji et al., 2013), and the seedlings with the third leaf fully expanded were treated with different Pi conditions. Plants were grown in an artificial climate chamber with a 14 h light/10 h night photoperiod, 30 °C /24 °C (day/night) temperature, and the humidity was controlled at 60%.

RNA extraction, cDNA synthesis, and RT-qPCR
Roots or shoots of rice plants were sampled and frozen in liquid nitrogen. Total RNA was extracted from rice roots and shoots using TRIzol™ reagent (Invitrogen). Reverse transcription was performed using ReverTra Ace ® qPCR RT Master Mix with gDNA Remover (Toyobo). Quantitative reverse transcription-PCR (RT-qPCR) was performed with AceQ ® qPCR SYBR Green Master Mix (Vazyme) on the QuantStu-dio™ 6 Flex Real-Time PCR System (Applied Biosystems). All experiments were carried out strictly according to the manufacturers' protocols. OsActin1 (LOC_Os03g50885) and OsHistone H3.3 (LOC_Os06g04030) were used as internal controls, and all the qPCR figures in this study were plotted with OsActin1 as an internal control and presented as 2 -ΔCT . All primers used for RT-qPCR are listed in Supplementary Table S2.

Tissue localization analysis
Histochemical analysis was performed as described in Ai et al. (2009). Different tissues of ProWRKY10:GUS transgenic plants were sampled for β-glucuronidase (GUS) staining. Rice tissues were embedded in 5% agar and cut into sections of 100 μm thickness using a Leica VT1200S (Leica). Tissues and sections were observed and photographed using a microscope (Zeiss).

Subcellular localization analysis
The full-length ORF of WRKY10 without a stop codon (Supplementary Table S3) was cloned into the pSAT6AEGFP-N1 vector. The plasmid of WRKY10-GFP and green fluorescent protein (GFP) alone were transformed into rice protoplasts using the polyethylene glycol (PEG)-mediated method as described in Dai et al. (2022). Nuclear localization signal (NLS)-mCherry was used as a nuclear control; the NLS sequence was synthesized by Genscript according to the methods described by Ji et al. (2016). Confocal images were photographed using a TCS SP8 X confocal laser scanning microscope (Leica) after incubation in the dark at 28 °C for 12-15 h. The excitation/emission wavelength of eGFP is 488 nm/498-540 nm, and that of mCherry is 552 nm/600-640 nm.

Measurement of Pi and total P concentration in rice
Measurement of Pi was performed as described by Zhou et al. (2008). Briefly, the fresh samples were ground in liquid nitrogen and extracted with 1 ml of 10% (w/v) perchloric acid and then diluted with 9 ml of 5% (w/v) perchloric acid. After the reaction between extract and working solution [0.4% (w/v) ammonium molybdate dissolved in 0.5 M H 2 SO 4 (solution A) and 10% (w/v) ascorbic acid (solution B)], the absorbance at 820 nm was measured using the Perkin-Elmer EnSight system. For total P measurement, different tissues were dried at 80 °C to constant weight. About 0.05 g of dry power was digested in 3 ml of an acid mix (nitric acid:perchloric acid=85:15, v/v). The digested liquid was fixed with ultra-pure water. The diluted liquid was filtered and measured using inductively coupled plasma optical emission spectroscopy (ICP-OES; Agilent 710).

Isotope labeling with 32 P uptake assay
Pi uptake assay was performed as described previously (Chang et al., 2019). Briefly, wild-type and wrky10 or pht1;2 mutant plants were treated with Pi-sufficient (90 μM) or Pi-deficient (1 μM) conditions until the sixth leaf blades were fully expanded. Roots were pre-treated with solution I (2 mM MES and 0.5 mM CaCl 2 , pH 5.5) for 10 min before being transferred to solution II (nutrient solution with 100 μM NaH 2 PO 4 containing 8 μCi l -1 of 32 P), and then cultured with solution II for 3, 8, and 24 h. After washing with deionized water, rice roots were transferred to icecold solution III (2 mM MES, 0.5 mM CaCl 2 , and 100 μM NaH 2 PO 4 , pH 5.5). Shoots and roots were harvested and digested by perchloric acid and hydrogen peroxide in 70 °C water baths until the liquid was fully clear. Subsequently, 0.2 ml of supernatant was mixed with 3 ml of scintillation cocktail (PerkinElmer Ultima Gold LLT), and radioactivity was measured with a scintillation counter (Beckman Coulter LS6500).

Yeast one-hybrid (Y1H) assay
Y1H assay was performed using the Matchmaker ® Gold Yeast One-Hybrid Library Screening System Kit (Clontech Biotechnology). Various fragments with a W-box site (Supplementary Table S3) were synthesized by Genscript and cloned into the pAbAi vector. The above constructs were linearized and transformed into Y1HGold yeast strain as the bait reporter strain. Then the yeast expression vector with empty vector or WRKY10 were each transformed into the bait reporter strain for Aureobasidin A (AbA) screening. The performance was carried out strictly according to the manufacturer's protocols.

Electrophoretic mobility shift assay
The full-length coding sequence (CDS) of WRKY10 was cloned into pMal-c5x (NEB). The empty vector and maltose-binding protein (MBP)-WRKY10 recombinant vector were each transformed into Escherichia coli BL21. Fusion proteins were purified using Amylose Resin (NEB). Protein concentrations were detected using the BCA Protein Assay Kit (Solarbio). The synthesis of probes and EMSA performance was as described in Zhang et al. (2021). All primers and probes are listed in Supplementary Table S3.

Chromatin immunoprecipitation assay
ChIP assay was performed using the EpiQuik™ Plant ChIP Kit (Epigentek) according to the manufacturer's protocols. Briefly, 1.0 g of rice roots of the wild type and ProActin1:WRKY10-FLAG were harvested for fixing with 1% formaldehyde, and the chromatin was sheared by sonication (Bioruptor Pico) to obtain DNA fragments. Anti-FLAG monoclonal antibodies (Invitrogen) were used for immunoprecipitation. The immunoprecipitated genomic DNA fragments were used for qPCR. Enrichment was calculated according to the ratio of immunoprecipitation to input. The primers are listed in Supplementary Table S3.

Yeast two-hybrid (Y2H) assay
The full-length ORF of MYB1 and truncated WRKY10 were cloned into pAD-GAL4-2.1 and pBD-GAL4-Cam, respectively. pAD, pBD, MYB1-pAD, and WRKY10-N-Δ60-pBD were transformed into YRG2 according to the pairwise combination of bait and prey, respectively. Transformants were selected on synthetic dextrose (SD) medium lacking leucine (L) and tryptophan (W). Yeast transformants from SD/-W were spotted onto solid SD/-L/-W or SD/-L/-W/-histidine (H) medium for observation and photographed after 3 d. The experiment was carried out strictly according to the manufacturer's protocols.

Cell-free degradation assay
The wild-type plants grown under +P and -P conditions were harvested and ground in liquid nitrogen for protein extraction. Total proteins were extracted in degradation buffer containing 25 mM Tris-HCl (pH 7.5), 10 mM NaCl, 10 mM MgCl 2 , 4 mM phenylmethylsulfonyl fluoride (PMSF), 5 mM DTT, and 10 mM ATP. The purified MBP-WRKY10 and MBP were incubated with plant protein at 28 °C for various times without or with MG132. Anti-MBP monoclonal antibodies (NEB) and ALEXA FLUOR 680 (Invitrogen) were used for western blot. Images were photographed using the Odyssey Imaging System (Li-Cor Biosciences).

WRKY10 inhibits Pi uptake
Mutants and overexpression lines of WRKY10 were generated and identified, and designated as wrky10 and WRKY10-Ox, respectively (Supplementary Figs S1, S2A). Wild-type, wrky10, and WRKY10-Ox plants were subjected to both high (HP, 90 µM) and low (LP, 1 µM) Pi treatments in a hydroponic system. At the seven-leaf stage, the rice seedlings were used for evaluating the growth performance and P accumulation. Under both Pi levels, neither mutation nor overexpression of WRKY10 led to an obvious alteration in growth performance ( Fig. 1A; Supplementary Fig. S3A). Under HP conditions, WRKY10-Ox lines had a level of P concentration comparable with that of the wildtype plants ( Supplementary Fig. S3B), whereas wrky10 mutants showed significantly enhanced P accumulation (Fig. 1B). The increase in P concentration observed in wrky10 was only evident in the shoot when the external Pi level was low (Fig. 1B).
To investigate whether the increased P accumulation in wrky10 mutants was attributed to enhanced Pi uptake, a shortterm Pi uptake assay with radioactive 32 P was performed. Pireplete and Pi-deficient plants were exposed to a solution containing 32 P. At all time points (3, 8, and 24 h), the Pi-replete but not the Pi-deficient wrky10 mutants had significantly higher Pi uptake as compared with wild-type plants (Fig. 1C). These results indicate that WRKY10 inhibits Pi uptake in a Pidependent manner. The elevated P accumulation in LP wrky10 plants (Fig. 1B) probably occurred during the pre-treatment (before LP treatment) when sufficient Pi was supplied.

WRKY10 is responsive to Pi starvation stress and encodes a nucleus-localized protein
The transcriptional response of WRKY10 to Pi starvation stress was investigated by a time-course analysis via RT-qPCR. A PSI marker gene, IPS1, was induced in both root and shoot by a 5 d Pi starvation treatment, and this induction reached the maximum level at days 7 and 10 in root and shoot, respectively ( Fig. 2A, B). In either shoot or root, IPS1 expression was significantly suppressed by a 1 d replenishment of Pi, and fell to the basal level after one further day of Pi supply ( Fig. 2A,  B). In contrast to IPS1, WRKY10 tended to be transcriptionally suppressed by Pi starvation. In root, WRKY10 was downregulated by Pi starvation after 5 d and this down-regulation was maintained until day 10 ( Fig. 2A). In the shoot, the downregulation of WRKY10 occurred earlier (at day 1) than that in the root (Fig. 2B); however, this suppression of WRKY10 expression fluctuated at day 7 when its expression level was comparable under the two Pi regimes. Nevertheless, WRKY10 expression was markedly induced by a 1 d replenishment of Pi in both root and shoot, and decreased to the basal level after one further day of Pi resupply ( Fig. 2A, B).
On the other hand, the subcellular localization of WRKY10 was analyzed by using PEG-mediated rice protoplast transformation. The results showed that GFP alone (serving as the positive control) was localized to almost all the intracellular compartments, including the cytoplasm and nucleus (Fig.  2C). In contrast, the signal emitted by the WRKY10-GFP fusion protein completely overlapped with that emitted by the mCherry reporter which is fused with an NLS (NLS-mCherry; Fig. 2C). These results indicate that WRKY10 encodes a nucleus-localized TF.

WRKY10 shows preferential tissue localization
To investigate the tissue localization of WRKY10, transgenic rice plants were generated via transforming a construct containing the GUS reporter gene which was driven by a putative promoter of WRKY10 with a length of 2200 bp. A histochemical analysis was then performed with the ProWRKY10:GUS plants. In crown root, WRKY10 expression was not observed at the root tip, similar to what was found for several PHT1 genes (Ai et al., 2009;Jia et al., 2011;X.F. Wang et al., 2014;Chang et al., 2019), whereas its expression was detectable at the middle part of the crown root, and was further enhanced in the basal part ( Fig. 3A-C). In the crown root base, WRKY10 was expressed in the sclerenchyma cell layer, cortex, pericycle, and vascular parenchyma, but not in the epidermal cells, exodermis, or endodermis (Fig. 3D); in the middle part of the crown root, WRKY10 expression was restricted to the inner cell layers of the cortex and the emerged lateral root (Fig. 3E). In the leaf sheath, WRKY10 expression was detected in almost all the cell types, with a higher level in the vasculature (Fig. 3F, G); in the leaf blade, WRKY10 was only expressed in the vascular tissue (Fig. 3H, I).

Expression of the WRKY10-Virus Protein 16 (VP16) fusion leads to increased P accumulation
Given that WRKY10 is a negative regulator of Pi uptake and accumulation (Fig. 1B, C), it could be postulated that WRKY10 exerts its function by repressing gene(s) with a positive role in Pi uptake and accumulation. To test this possibility, the activation domain of VP16, which could turn repressors into activators (Sadowski et al., 1988;Kong et al., 2016), was fused to the C-terminus of WRKY10. Three independent transgenic lines (WRKY10-VP16-2, WRKY10-VP16-10, and WRKY10-VP16-15) were selected for further investigation. WRKY10-VP16-10 and WRKY10-VP16-15 showed a high expression level of WRKY10-VP16, whereas WRKY10-VP16-2 gave rise to no expression of the fusion, and was thus used as an additional negative control (NC; Supplementary  Fig. S2B).
Under HP conditions, the WRKY10-VP16-10 and WRKY10-VP16-15 plants showed typical Pi toxicity symptoms as evidenced by arrested growth as well as chlorosis and necrosis of old leaf tips (Fig. 4A, B; Chiou et al., 2006;Hu et al., 2011). As expected, the P concentration in the root and shoot of WRKY10-VP16-10 and WRKY10-VP16-15 but not NC was significantly increased as compared with that of the wild-type plants (Fig. 4C). Notably, this increase in P accumulation was similar to but more evident than (>1% of DW in the shoot) that found in wrky10 mutants (Figs 1B, 4C), suggesting that WRKY10 functions as a transcriptional repressor of Pi uptake and accumulation. Under LP condition, an increase in P concentration was observed only in the shoot of WRKY10-VP16-10 and WRKY10-VP16-15, also similar to that found in wrky10 mutants (Figs 1B, 4C).

WRKY10 suppresses the expression of OsPHT1;2 via binding to its promoter
To investigate whether WRKY10 affects Pi uptake through direct regulation of PHT1 genes, the expression of PHT1 genes was examined in the mutants and overexpression lines of WRKY10. All the PHT1 genes responded to Pi starvation stress as expected irrespective of the genotypes-OsPHT1;1 tended to be constitutively expressed or even slightly downregulated by Pi starvation, whereas OsPHT1;2/1;3/1;4/1;6/1 ;8/1;9/1;10 were induced, to different extents, by Pi starvation (Supplementary Fig. S4; Fig. 5). In addition, three out of eight PHT1 genes tested, OsPHT1;2, OsPHT1;3, and OsPHT1;10, were up-regulated in Pi-replete but not Pi-deficient wrky10 mutants as compared with wild-type plants (Fig. 5A), suggesting that WRKY10 inhibits the expression of these three PHT1 genes in a Pi-dependent manner. The expression of PHT1 genes in the WRKY10-VP16 plants was also examined. Consistent with what was found in wrky10 mutants, the expression of OsPHT1;2, OsPHT1;3, and OsPHT1;10 was also enhanced under Pi-replete conditions, but to a larger extent (Fig. 5B).
WRKY TFs regulates their targets by binding to the W-box in the promoters (Rushton et al., 2010). OsPHT1;10 does not carry any W-box in its putative promoter region (Supplementary Fig. S5), thus it is less likely that OsPHT1;10 is a direct target of WRKY10; additionally, OsPHT1;3 and OsPHT1;10 have a much lower abundance than OsPHT1;2 under Pi-replete conditions even when they were up-regulated in WRKY10-VP16 plants ( Fig. 5; Secco et al., 2013;X.F. Wang et al., 2014;Chang et al., 2019). Based on these facts, we reasoned that OsPHT1;2 could be the major contributor to the elevated Pi uptake in wrky10 mutants and WRKY10-VP16 plants. Consequently, we first tested the potential physical interaction between WRKY10 and OsPHT1;2 through Y1H assay. Two fragments in the proximal promoter region of OsPHT1;2 containing the W-box are designated as Fragment 1 (F1) and Fragment 2 (F2) (Fig. 6A,  B). The yeast cells transformed with WRKY10 and F1 showed suppressed growth compared with those transformed with the empty vector (EV; negative control) and F1 when 400 ng ml -1 Fig. 4. Expression of the fusion of WRKY10 and Virus Protein 16 (VP16) leads to increased P accumulation. Rice seeds were germinated in sterilized water and supplied with 1/2 strength Kimura B solution until the third leaf blades were fully expanded, and then treated with HP (90 μM) and LP (1 μM) until the sixth leaf blades were fully expanded; four biological repeats are set for each treatment. (A) Phenotype of wild-type (WT) and WRKY10-VP16 transgenic plants grown under HP (left) and LP (right) conditions. Scale bars=10 cm. (B) Phenotype of fourth leaf blades of WT and WRKY10-VP16 under HP (left) and LP (right) conditions. (C) Total P concentration in shoot and root under HP (left) and LP (right) conditions. All data are plotted with boxwhisker plots: the whiskers represent maximum and minimum values, and boxes represent the upper quartile, median, and lower quartile. The results shown are from four biological replicates. Data significantly different from the corresponding controls are indicated (*P<0.05, **P<0.01; Student's t-test). NC, negative control.
AbA was supplied in the medium, suggesting that WRKY10 can bind to the W-box in F1 and exerts transcriptional repression activity in yeast (Fig. 6C). In contrast, it seems that WRKY10 displays transcriptional activation activity in yeast as well, since the yeast cells transformed with WRKY10 and F2 showed normal growth while the growth of the cells transformed with EV and F2 was largely inhibited when 800 ng ml -1 AbA was added in the medium (Fig. 6C). Nevertheless, the results obtained through the Y1H system demonstrate that WRKY10 binds to both W-box elements in the OsPHT1;2 promoter. To further investigate the physical interaction between WRKY10 and OsPHT1;2, EMSA and ChIP-qPCR were performed. Both experimental systems validated the interaction between WRKY10 and the two copies of the W-box in the PHT1;2 promoter ( Fig.  6D, E). All these results demonstrated that OsPHT1;2 is a direct target of WRKY10.

OsPHT1;2 is involved in Pi uptake and its mutation counteracts the enhanced Pi accumulation in wrky10 mutants
Consistent with our previous results, mutation of OsPHT1;2 led to decreased Pi accumulation under LP but not HP condition ( Supplementary Fig. S6; Fig. 7A) . To investigate whether the decreased Pi accumulation in ospht1;2 mutants ( Fig. 7A) results from a defect in Pi uptake, a radioactive 32 P-labeled Pi uptake assay was performed. Plants grown under HP or LP conditions were subjected to an equal amount of Pi (100 μM Pi) labeled with 32 P. The Pi uptake was comparable between Pi-replete ospht1;2 mutants and wild-type plants at each time point (3, 8, and 24 h); in contrast, the Pi uptake of ospht1;2 mutants subjected to LP stress was significantly decreased at 3 h and 8 h compared with that of wild-type plants (Fig. 7B). The difference in Pi uptake in ospht1;2 mutants was absent after 24 h (Fig. 7B), suggesting that OsPHT1;2 is also rapidly repressed by Pi resupply, similar to that found in ospht1;3 mutants (Chang et al., 2019). Nevertheless, all these results demonstrate that OsPHT1;2 is involved in Pi uptake at least under LP conditions.
To further investigate whether OsPHT1;2 is the cause for the elevated Pi accumulation in wrky10 mutants, a cross between wrky10 and pht1;2 mutants was performed (Supplementary Fig. S7), and then the F2 progeny were used for evaluating Pi accumulation. Mutation of OsPHT1;2 in the background of wrky10 (wrky10 pht1;2 double mutant) led to a decrease in Pi accumulation to a level comparable with that in wildtype plants (Fig. 7C). These results indicate that OsPHT1;2 functions downstream of WRKY10 and is responsible for the increased Pi accumulation in wrky10 mutants.

WRKY10 protein is degraded by the 26S proteasome system in response to Pi starvation
The abundance of WRKY TFs is known to be regulated at multiple levels (e.g. the transcriptional and post-translational levels). Since WRKY10 is negatively regulated by Pi starvation  (C) WRKY10 binds to the PHT1;2 promoter in yeast. F1 and F2 were each integrated into yeast genomic DNA as bait vectors, then WRKY10 or empty vector (EV) were transformed into yeast containing the bait vector. OD 600 values of yeast cells grown in SD/-Leu medium were set as 10 -1 , 10 -2 , and 10 -3 . A 4 µl aliquot of diluted suspension was spotted on SD/-Leu medium with different concentrations of Aureobasidin A (AbA). (D) EMSA to detect the binding of WRKY10 to the PHT1;2 promoter in vitro. Each biotin-labeled probe was incubated with MBP or MBP-WRKY10 protein. Excess unlabeled probes (cold probe or mutant probe) were added to compete with biotin-labeled probes. The WRKY10-DNA complex (bound probes) and free DNA probes (free probes) are indicated by black arrows. (E) ChIP-qPCR assay to determine the binding of WRKY10 to the PHT1;2 promoter in vivo. Rice seeds of the wild type (WT) and ProActin1:WRKY10-FLAG were germinated in sterilized water and supplied with 1/2 strength Kimura B solution. The root was harvested for ChIP assay. Enrichment of each site was quantified using qPCR analysis. Data significantly different from the corresponding controls are indicated (*P<0.05, **P<0.01; Student's t-test). at the transcriptional level ( Fig. 2A, B), it would be important to examine whether WRKY10 protein is regulated at the post-translational level in a Pi-dependent manner. Thus, a cell-free protein degradation assay was performed. The recombinant MBP-WRKY10 was stable when incubated with the HP total protein extract, while it was markedly degraded when incubated with the LP total protein extract (Fig. 8). In contrast, MBP was rather stable when incubated in either HP or LP total protein extract (Fig. 8). In addition, the degradation of WRKY10 was inhibited by MG132, a 26S proteasome inhibitor (Fig. 8), indicating that Pi starvation induced the 26S proteasome-dependent degradation of WRKY10.

Discussion
WRKY10 negatively regulates OsPHT1;2 expression in a Pi-dependent manner Mukatira et al. (2001) provided the first evidence in Arabidopsis that PSI genes (including a PHT1 member) may be under negative regulation when Pi is sufficient. In our previous work, we validated this possibility in rice by showing that OsPHT1;2 and one of its homologs (OsPHT1;8) are transcriptionally suppressed by OsMYB1 (Gu et al., 2017). In spite of these findings, the molecular evidence for the TF(s) physically interacting with and negatively regulating PHT1 genes is still lacking. In the present work, we demonstrated that WRKY10 suppresses Pi uptake under Pi-sufficient conditions by negatively regulating OsPHT1;2 expression via binding to its promoter (Figs 1C,5,6). Notably, OsPHT1;2 protein abundance is also negatively regulated (by OsNLA1) under Pi-sufficient conditions (Yue et al., 2017). Our results and reported findings suggest that OsPHT1;2 is a major target for inhibiting Pi uptake, and that transcriptional repression of OsPHT1;2 is an indispensable event in this process. Intriguingly, another OsPHT1;2 homolog, OsPHT1;1, which is not responsive to Pi availability at the transcriptional level, is positively regulated by a pair of WRKY TFs, OsWRKY21 and OsWRKY108; the maintenance of basal OsPHT1;1 expression by OsWRKY21/108 is required for Pi uptake under Pi-sufficient condition (Sun et al., 2012;Zhang et al., 2021). Thus, the OsWRKY21/108-OsPHT1;1 and WRKY10-OsPHT1;2 regulatory modules represent a flexible and complementary system of the Pi signaling network in rice: under Pi-replete conditions, the activation (by the OsWRKY21/108-OsPHT1;1 module; Zhang et al., 2021) and repression (by the WRKY10-OsPHT1;2 module; Figs 6-8) of Pi uptake occur simultaneously to maintain P homeostasis. These findings suggest that the functional divergence of PHT1 members (e.g. OsPHT1;1 and OsPHT1;2) is, to a large extent, attributed to the distinct mechanisms monitoring their transcription in response to the fluctuating Pi availabilities in the environment. Interestingly, overexpression of WRKY10 did not lead to any alteration in Pi accumulation (Supplementary Fig. S3). One possible explanation for this is that the transcriptional suppressing activity of WRKY10 may require the coordination of an unknown protein, but the endogenous abundance of this unknown protein is not sufficient to confer transcriptional activity on the WRKY10 derived from the transgenic process. In future work, it would be useful to identify the potential protein(s) interacting with WRKY10 to monitor its transcriptional activity.
Although OsPHT1;2 is the major contributor to the increased Pi accumulation in wrky10 mutants (Fig. 7), it should be noted that two other PHT1 homologs barely expressed under Pi-replete conditions, OsPHT1;3 and OsPHT1;10, were also up-regulated in wrky10 mutants and WRKY10-VP16 plants under HP but not LP conditions (Fig. 5). This altered expression profile of PHT1 genes is reminiscent of that found in a transgenic rice line overexpressing a PSI gene encoding a RING-type E3 ligase, OsPIE1 (Pi-starvationinduced E3 Ligase; Yang et al., 2018). In addition, overexpression of OsPIE1 or mutation of WRKY10 specifically suppressed the expression of OsSPX2 but not OsSPX1 ( Supplementary  Fig. S8) (Yang et al., 2018). These unique regulations of downstream PSI genes shared by OsPIE1 overexpressors and wrky10 mutants suggest that OsPIE1 and WRKY10 may be involved in the same subcascade of the Pi signaling network in rice. Given that WRKY10 abundance is negatively regulated by the ubiquitin-26S proteasome (Fig. 8), one tempting assumption is that OsPIE1 mediates the ubiquitination and degradation of WRKY10 protein under Pi starvation conditions. In future work, it would be interesting to further investigate the potential genetic interaction between WRKY10 and OsPIE1 or other unknown E3 ligase(s) regarding their involvement in regulating Pi uptake.
On the other hand, the crown root is the major type of root comprising the root system of rice plants which is vital for the uptake of nutrients (Coudert et al., 2010). In rice root, the two cell layers with a casparian strip (i.e. exodermis and endodermis) have been shown to play a vital role for the uptake of several mineral nutrients as well as toxic elements (Sasaki et al., 2016); however, it cannot be excluded that other cell layers (e.g. sclerenchyma cell layer and cortical cells) may contribute to nutrient uptake since they represent integral parts of the predestined route of short-distance transport, which starts from the epidermis, then to the exodermis, sclerenchyma cell layer, cortex, endodermis, and finally to the stele (Sasaki et al., 2016;Huang et al., 2022). Interestingly, WRKY10 is expressed in almost all the cell types of the crown root except the epidermis, exodermis, and endodermis (Fig. 3). TFs and their direct target genes are usually but not always expected to be expressed in the same cell types. Unfortunately, the tissue/cellular localization of OsPHT1;2 remains obscure. In our previous work, we showed that OsPHT1;2 is expressed in root stele by using transgenic rice lines harboring the GUS reporter gene driven by a putative promoter fragment of OsPHT1;2 (Ai et al., 2009). Zhang et al. (2014) challenged this result by using mRNA in situ hybridization and claimed that OsPHT1;2 expression was detected in root epidermal cells but not in the stele. However, the signal detected by Zhang et al. (2014) was actually the sclerenchyma cell layer [the third cell layer from the outermost layer (epidermis)]. In addition, a combination of laser microdissection and microarray analysis showed that the OsPHT1;2 transcript was most abundant in a mixed tissue comprised of three cell layers, namely the epidermis, exodermis, and sclerenchyma layers (Supplementary Fig. S9; https://ricexpro.dna. affrc.go.jp/). Thus, to investigate whether the regulation of WRKY10 on OsPHT1;2 is a cell-autonomous or non-cellautonomous event, a re-examination of OsPHT1;2 tissue/cellular localization by other approaches (e.g. targeted insertion of an epitope tag into the OsPHT1;2 locus in the rice genome followed by immunostaining analysis; Gu et al., 2016;Dai et al., 2022) is required.
Notably, it seems that the role of WRKY10 in P homeostasis is also obvious in the shoot, since the increase in P concentration is more evident in the shoots than that in the roots of wrky10 mutants (Fig. 1B). Indeed, the percentage of P translocated to the shoot was significantly increased in wrky10 mutants ( Supplementary Fig. S10), suggesting that the WRKY10-OsPHT1;2 module might be involved in P distribution between the root and shoot in addition to Pi up-take. Unraveling the underlying physiological mechanism also requires information on the cellular localization of OsPHT1;2. Nevertheless, in this work, we provide several lines of evidence that WRKY10 inhibits Pi uptake through suppressing OsPHT1;2 expression via binding to its promoter.
The wrky10 myb1 double mutant does not show additive or synergistic effect on Pi accumulation In our previous work, we demonstrated that a MYB TF in rice, OsMYB1, negatively regulates Pi uptake by suppressing the expression of OsPHT1;2 and OsPHT1;8 (Gu et al., 2017). To investigate whether OsMYB1 and WRKY10 regulate OsPHT1;2 expression coordinately or independently, a wrky10 myb1 double mutant was generated by crossing wrky10 and myb1 (Supplementary Fig. S11). The Pi accumulation in wrky10 myb1 was significantly higher than that in wild-type plants, but was not altered as compared with its parental lines, namely wrky10 and myb1 ( Supplementary Fig. S12A). Given that WRKY10 directly regulates OsPHT1;2 expression by binding to its promoter (Figs 5, 6), two possibilities could explain the lack of additive or synergistic effect on Pi accumulation in wrky10 myb1: (i) OsMYB1 indirectly regulates OsPHT1;2 expression by activating WRKY10 expression; or (ii) OsMYB1 and WRKY10 coordinately regulate OsPHT1;2 expression. To test these two possibilities, the expression of WRKY10 was first examined in myb1 mutants. No alteration in WRKY10 expression in myb1 mutants was found ( Supplementary Fig.  S12B), indicating that OsMYB1 is not an upstream regulator of WRKY10. Subsequently, the potential physical interaction between WRKY10 and OsMYB1 was tested via a Y2H system; however, no interaction was detected either ( Supplementary  Fig. S12C). These results suggest that WRKY10 and OsMYB1 coordinately regulate OsPHT1;2 expression in an unknown manner without physical interaction, and/or their physical interaction cannot be detected in yeast. Nevertheless, it would be of interest to investigate how WRKY10 and OsMYB1 coregulate the expression of OsPHT1;2 in future work.

A working hypothesis
Our working hypothesis for the regulation of P homeostasis via the WRKY10-OsPHT1;2 module is shown in Fig. 9. Under Pi-sufficient conditions, WRKY10 expression is relatively high ( Fig. 2A, B), and it may suppress OsPHT1;2 expression both directly and indirectly. WRKY10 suppresses the transcription of OsPHT1;2 via direct binding to its promoter (Figs 5, 6); additionally, WRKY10 is required for maintaining the basal expression of OsSPX2 which indirectly suppresses OsPHT1;2 expression by impeding the function of OsPHR2 via protein-protein interaction with OsPHR2 (Puga et al., 2014;. In Pi-replete wrky10 mutants, the suppressive effect of WRKY10 on OsPHT1;2 transcription is largely abolished; thus, the repression of Pi uptake is alleviated, leading to enhanced Pi uptake and accumulation (Fig. 1). Upon Pi deficiency, WRKY10 abundance is suppressed at both the transcriptional and post-translational level (Figs 2A,  8). The degradation of WRKY10 protein is dependent on the 26S proteasome (Fig. 8) and might be mediated by PIE1 and/ or unknown E3 ligase(s). Due to the decrease in WRKY10/ WRKY10 abundance, the suppressive effect of WRKY10 on OsPHT1;2 (either direct or indirect) is alleviated, leading to enhanced Pi uptake. The inhibition of WRKY10 abundance in response to Pi starvation at both the transcriptional ( Fig. 2A, B) and post-translational (Fig. 8) level further demonstrates the importance of the WRKY10-OsPHT1;2 module in maintaining P homeostasis.

Supplementary data
The following supplementary data are available at JXB online. Fig. S1. Identification of wrky10 mutant plants. Fig. S2. Identification of Pro35S:WRKY10 and Pro35S:WRKY10-VP16 plants. Fig. S3. Physiological analysis of wild-type and WRKY10 overexpression plants. Fig. S4. Expression of PHT1 genes in root of wrky10 mutant plants.  Table S1. Primers used for constructs for generating transgenic plants. Table S2. Primers used for RT-qPCR analysis. Table S3. Primers used for constructs for subcellular location, Y1H, EMSA, and ChIP-qPCR assay.
of Michigan) for providing us with the vectors for the CRISPR/Cas9 system and subcellular localization analysis.

Author contributions
MG: conceptualization and design; SCW: performing most of the experiments; TTX, MC, and LYG: field management and assisting in the physiological analysis; XLD, HYQ, and HHL: performing hydroponic culture and rice transformation; ZYH: constructing part of the expression vectors; JZ and HHL: performing preliminary analysis of the mutant phenotype; SCW and MG: data analysis and writing the article; GHX: providing critical comments and revisions to the manuscript.